Human alpha-1-proteinase inhibitor (A-1-PI), which is a serine protease inhibitor and which in vivo inhibits the activity of neutrophil elastase, undergoes guanidine-HCl (Gu) induced biphasic unfolding with an intermediate state in 1.5 M Gu. Previously, we demonstrated that this intermediate state can consist of one or more folding intermediates. Size exclusion-HPLC in 1.5 M Gu shows that an equilibrium distribution of monomeric and polymeric intermediates forms over 2 h at 25 deg C. These polymeric intermediates are primarily dimeric, trimeric, and tetrameric with some higher polymers and are of a loop-sheet nature (i.e the reactive site loop, which normally targets the active site of elastase, is inserted between the central strands of a beta sheet region on another A-1-PI molecule). Folding by dilution from 1.5 M Gu gives levels of active monomer and inactive polymer that reflect those of the intermediates. Kinetic studies demonstrate that this equilibrium state is on the folding pathway and that folding from the fully unfolded state is under kinetic control thereby allowing the formation of the less stable but active monomer. Since starting in January, Dr. Marszal has discovered a much slower, higher order aggregation that occurs at 25 deg C over a period of days after the relatively rapid polymerization observed in 1.5 M Gu. This slower aggregation is observed both for the intermediates in 1.5 M Gu and for monomer and polymers formed from folding from 1.5 M Gu although the latter is slower. The polymer that forms elutes in the void volume of tandem TSK 3000 columns. Dr. Marszal has demonstrated that this high-order aggregate may be dissociated to monomer with high Gu. She is in the process of characterizing the nature of this high-order aggregate, which may not be of a loop-sheet nature. An interesting possibility is that the rapid polymerization in 1.5 M Gu may relate to the secretion defect in patients with mutant A-1-PI and the slower high-order aggregation to the formation of inclusion bodies of this mutant A-1-PI in the hepatocytes, the site of synthesis. In determining the specific inhibitory activity (potency) of samples of A-1-PI, Dr. Marszal utilized trypsin as the target protease, for which she accurately measured the concentration of the active enzyme with a chromogenic active site titrant. She has observed that at low trypsin and low A-1-PI concentrations, the binding of A-1-PI is not stoichiometric thereby indicating that the inhibition constant is lower than assumed. Thus, she plans to determine this constant in order to establish optimal conditions for determining the concentration of active A-1-PI thereby allowing an accurate redetermination of the active concentration of the CBER A-1-PI Reference Standard, which currently is estimated to have approximately 75% of its originally labeled potency. The stability of this standard will be monitored in terms of potency.